Genotyping diptera using amplified fragment length polymorphism (AFLP): development of a genetic marker system for species in the families Calliphoridae and Sarcophagidae

Abstract

Amplified Fragment Length Polymorphism (AFLP) analysis of entomological material provides a means of obtaining a reproducible DNA profile in a relatively short period of time. AFLP profiles were obtained using larval samples from Cochliomyia macellaria, Phormia regina and Sarcophaga bullata. Genomic DNA was isolated using the DNeasy Tissue Kit (Qiagen, Valencia, CA), double-digested by two restriction endonucleases (EcoRI and MseI) and ligated to oligonucleotide adapters. Two consecutive PCR reactions (preamplification and selective amplification) were performed using a modification of the AFLP protocol described by Gibco (Invitrogen, Rockville, MD). The DNA fragments were separated by capillary electrophoresis using the CEQ 8000 DNA Fragment Analyzer. Species specific profiles were developed for all three species. The results indicate that the AFLP technique is a viable and valuable technique for identification of entomological material.